HIV protection by ZFN-based disruption of CCR5 gene in Hematopoietic stem cells

描述（由申请人提供）：使用锌指核酸酶和TALEN的基因组编辑已经成为一种有前途的技术，用于在靶基因中诱导局部序列突变。由于CCR 5是HIV的主要共受体，研究正在进行中，以破坏T细胞和CD 34+造血干细胞中的基因，以保护细胞或其后代免受病毒感染。到目前为止，病毒和质粒载体已用于递送ZFN/TALEN。然而，这导致核酸酶的长期表达，这可能是潜在有害的，因为越来越多的研究已经记录了许多工程化ZFN的剂量依赖性毒性，推测是由于在脱靶位点产生非预期的双链断裂。从mRNA瞬时表达ZFN/TALEN可以提供可行的替代方案，因为短期暴露于核酸酶足以引起靶基因的永久修饰。在该提议中，来自mRNA的ZFN或TALEN表达将被用作破坏CD 34+造血干细胞（HSC）中的CCR 5的策略。为了克服ZFN/TALEN mRNA递送至CD 34 + HSC的障碍，CD 34 + HSC是难以通过常规方法转染的细胞，我们将开发脂质纳米颗粒，其展示先前描述的DNA适体sgc 8，其结合在细胞上表达的蛋白酪氨酸激酶7。人源化BLT小鼠将用ZFN/TALEN修饰的CD 34 + HSPC重建，并测试对HIV攻击的保护。从研究中获得的知识可能会导致开发一种新的安全方法来利用ZFN/TALEN进行HIV基因治疗，甚至提供一种将分子直接递送到体内动员干细胞的潜在方法。 公共卫生相关性：定制锌指核酸酶（ZFN）基因干扰技术是一种新兴的基因治疗技术，在艾滋病等多种疾病的基因治疗中具有广阔的应用前景。该研究的主要目标是使用ZFN破坏CD 34+造血干细胞中的HIV共受体CCR 5作为基因治疗，以产生HIV抗性后代。由于ZFN的长期表达可能是有害的，我们计划开发一种用于细胞中瞬时mRNA表达的纳米颗粒递送平台，使得ZFN/TALEN仅在短时间内表达以发挥其CCR 5基因修饰活性。

DESCRIPTION (provided by applicant): Genome editing with Zinc Finger nucleases and TALENs has emerged as a promising technology for inducing localized sequence mutations in targeted genes. As CCR5 is the principal co-receptor for HIV, studies are underway to disrupt the gene in T cells and CD34+ Hematopoietic stem cells to protect the cells or their progeny from viral infection. So far, viral and plasmid vectors have been used for delivery of ZFNs/TALENs. However, this results in long-term expression of the nucleases, which may be potentially harmful, as increasing number of studies have documented dose-dependent toxicity of many engineered ZFN, presumably due to the generation of unintended double-stranded breaks at off-target sites. Transient expression of ZFNs/TALENs from mRNA may provide a feasible alternative as short term exposure to the nucleases is sufficient to cause permanent modification of the targeted gene. In this proposal, ZFN or TALEN expression from mRNA will be used as a strategy to disrupt CCR5 in CD34+ hematopoietic stem cells (HSCs). To overcome the hurdle of ZFN/TALEN mRNA delivery to CD34+ HSCs, which are difficult cells to transfect by conventional methods, we will develop lipid nanoparticles displaying a previously described DNA aptamer sgc8 that binds to Protein tyrosine kinase 7, expressed on the cells. Humanized BLT mice will be reconstituted with the ZFN/TALEN-modified CD34+ HSPCs and tested for protection from HIV challenge. Knowledge gained from the studies could lead to the development of a novel and safe approach to harness ZFN/TALEN for HIV gene therapy and even provide a potential method for direct delivery of the molecules to mobilized stem cells in vivo. PUBLIC HEALTH RELEVANCE: Gene disruption by tailored Zinc Finger nuclease (ZFN) is an emerging technology with promising applications in gene therapy of various diseases including AIDS. The main goal of the proposed research is to use ZFN to disrupt the HIV co-receptor CCR5 in CD34+ hematopoietic stem cells as gene therapy to generate HIV resistant progeny. As long-term expression of ZFN can be harmful, we plan to develop a nanoparticle delivery platform for transient mRNA expression in the cells so that ZFNs/TALENs are expressed only for a short time to exert their CCR5 gene modification activity.